Causal association of micronutrients and supplements with pressure ulcer: A Mendelian randomization study

Abstract Background Pressure ulcer (PU) is known to be associated with abnormalities of micronutrient status. However, to date, it is not clear whether a causal relationship exists between circulating levels of micronutrients and their supplementations and PU. Methods A two‐sample Mendelian randomization (MR) study was conducted using summary statistics from Genome‐Wide Association Studies (GWAS). Genetic instrumental variables (IVs) for 13 micronutrients were identified from a GWAS of 67 582 participants, IVs for supplement zinc were acquired from 18 826 cases and 44 255 880 controls, and IVs for PU were obtained from 663 PUs and 207 482 controls. The MR analysis was conducted using the MR base platform. The main analysis method was inverse variance weighted (IVW) analysis, supplemented by MR Egger, Weighted median, Weighted mode, and Simple mode analyses. Heterogeneity was assessed using Cochran's Q statistic for MR‐IVW and Rucker's Q statistic for MR‐Egger. Pleiotropy was determined by the MR‐Egger regression. Sensitivity analysis was conducted using the leave‐one‐out method, and publication bias was evaluated using funnel plots. Results Genetically predicted lower circulating zinc levels were found to be causally linked to the development of PU (OR = 0.758, 95%CI 0.583–0.987, P = 0.040). However, there was no significant evidence of a causal relationship between supplemental zinc intake and PU development (P > 0.05). Additionally, no causal association was observed between the other circulating micronutrients and the occurrence of PU. Furthermore, there was no indication of horizontal pleiotropy or heterogeneity among genetic variants (P > 0.05), and the robustness of the findings was confirmed through leave‐one‐out tests and funnel plots. Conclusions Our findings indicate a potential causal association between circulating zinc levels and decreased risk of PU. However, zinc supplementation did not demonstrate a significant reduction in the risk of PU. Further research is warranted to elucidate the underlying mechanisms through which zinc influences the pathogenesis of PU and evaluate the efficacy of zinc supplementation in the prevention and management of PU.


INTRODUCTION
Pressure ulcers (PU) are frequently observed in elderly individuals, particularly those with limited physical mobility or who are confined to bed and are influenced by a multitude of factors including the patient's underlying pathologies, the severity of the primary illness, presence of comorbidities, level of functional impairment, nutritional status, and availability of socio-emotional support. 1 A recent crosssectional study conducted in the United States observed a rising trend in both the annual prevalence and average annual cost of PU, 2 another study highlighted the potential for a growing burden on individual health and socioeconomics due to the aging population and increasing numbers of nursing home residents experiencing PU. 3 Prior research has established stress as a primary factor in the development of PU, with prolonged inactivity, sensory deficits, circulatory disorders, and malnutrition also recognized as significant risk factors. 4Recent findings indicate that PU results from sustained mechanical loading causing soft tissue deformation. 5However, the precise relationship between micronutrients, their supplements, and PU formation remains incompletely elucidated.
Micronutrients, such as trace elements and vitamins, play a significant role in the maintenance of overall health.Recent research has focused on the relationship between micronutrient levels and the development, progression, and treatment of PU.A study conducted by Schott M. 6 examining the risk of PU following a stroke revealed that deficiencies in certain nutritional factors, namely zinc, selenium, and copper, were associated with an increased likelihood of developing PU.
A recent clinical study conducted in China indicates a positive correlation between iron levels and the advancement of PU, potentially linked to the abnormal CD163/Homx-1 signaling pathway. 7Additionally, a retrospective cohort study conducted in Boston demonstrated a relationship between the vitamin D status of intensive care unit patients upon admission and the subsequent development of hospital-acquired PU. 8 A literature review published in 2017 showed that the clinical utilization of zinc and other micronutrient-containing supplements can be advantageous for individuals with PU, potentially enhancing outcomes, expediting healing processes, and mitigating complications. 9vertheless, the absence of substantial, high-quality clinical data on the impact of micronutrients on PU leaves uncertainties regarding the causal connection between circulating micronutrient levels, their supplementation, and the development of PU.
The Mendelian randomization (MR) method is a robust approach in epidemiological research, employing genetic variation to evaluate causal relationships between exposures (micronutrients and their supplements) and outcomes (PU). 10Through the random allocation of prenatal genetic alleles to offspring, MR demonstrates a superior ability to control for confounding variables that may introduce bias in observational studies, as well as mitigate issues related to reverse causation. 11To date, there has been a lack of comprehensive research investigating the causal relationship between micronutrient levels, their supplements, and the development of PU.Therefore, the objective of this study was to utilize a two-sample MR approach to elucidate the causal connection between circulating micronutrients and their supplements and the risk of PU.This research aims to provide clinical guidelines for the prevention and management of PU.

Data sources and study design
This research is reported according to the STROBE-MR (Supplementary material 1). 12As the GWAS dataset utilized in this study is openly accessible online and all participants underwent informed consent and ethical review in their original studies, no further ethical authorizations were deemed necessary.The initial step involved conducting MR analyses on micronutrients and PU to identify potential causal relationships.This was followed by MR analyses on the corresponding micronutrient supplements to assess their impact on the risk of PU.We conducted a comprehensive search of published Genome-Wide Asso- Base enhances the efficacy and dependability of hypothesis-driven methodologies. 13Consequently, our MR analysis was conducted utilizing the MR Base platform.Utilizing single nucleotide polymorphisms

Selection of IVs
To identify a sufficient number of SNPs strongly associated with micronutrients and their supplements, SNPs were selected at a genome-wide significance threshold (P < 5 × 10 −6 ). 15To mitigate the impact of linkage disequilibrium (LD) on the findings and ensure the selection of independent SNPs, the threshold for the LD parameter (r 2 ) was set at 0.001, with a genetic distance limit of 10 000 kb.The study evaluated the strength of correlations between IVs and exposure factors through the use of the F-statistic.SNPs with F-statistics exceeding 10 were selected to mitigate potential bias resulting from weak IVs. 16e R 2 and F-statistic for each SNP were calculated using the formu- respectively.

Statistical analysis
All Rucker's Q statistic. 18Additionally, we assessed visual asymmetries in the funnel plots. 19We used the MR-Egger method to assess horizontal multiplicity and conducted a leave-one-out analysis to investigate the potential impact of individual SNPs on the causal relationship between micronutrients, their supplements, and PU. 20P-value < 0.05 was considered for statistical significance, and the results were reported as the odd ratios (OR) with their 95% confidence intervals (CIs).

Effect of circulating micronutrients on PU
In the MR analysis examining the relationship between circulating micronutrients and PU, a total of 158 SNPs were ultimately included as IVs.All F-statistic values exceeded 10, ranging from 20.8 to 84.7, indicating a low likelihood of weak IV bias (Supplementary material 2).
The effects of 13 circulating micronutrients on PU were assessed using the IVW method as the primary approach, complemented by MR Egger, Weighted median, Weighted mode, and Simple mode analyses.Our study revealed that IVW analysis indicated a significant causal relation-ship between decreased zinc levels (OR = 0.758, 95%CI 0.583-0.987,P = 0.040) and PU (Figure 2A).Even the results of MR Egger analysis did not align with those of IVW, the consistency of results from MR analysis methods such as Weighted median, Weighted mode, and Simple mode with IVW results suggests the reliability of our findings (Figure 2B).
Detailed results of IVW, MR Egger, Weighted median, Weighted mode, and Simple mode analyses for the 13 micronutrients on PU can be found in Figure 2E and Supplementary material 3.There was a lack of heterogeneity observed in the MR analysis concerning decreased zinc levels and the occurrence of PU (MR-Egger Q = 4.582, P = 0.469; MR-IVW Q = 5.837, P = 0.4416).Furthermore, the absence of significant intercepts in the assessment of pleiotropy indicates the non-existence of pleiotropic effects (intercept = −0.12,P = 0.313).Additionally, the "leave-one-out" analysis confirmed the reliability of our MR analysis, as it remained unaffected by any individual SNP (Figure 2C), and the funnel plot did not exhibit any notable asymmetry (Figure 2D).

Effect of the supplement zinc on PU
In a prior investigation, a causal association was identified between circulating zinc levels and the occurrence of PU.A two-sample MR analysis was conducted to ascertain the causal effect of zinc supplements on PU.Eventually, 15 SNPs were utilized as IVs, with all F-statistic values surpassing 10, ranging from 21.0 to 33.5, indicating a low probability of weak IV bias (Supplementary material 4).In the MR analysis, it was determined that the IVW analysis did not reveal a significant causal association between supplement zinc and the development of PU (P > 0.05).This conclusion is supported by the data presented in Supplementary materials 5 and 6.Additionally, no evidence of heterogeneity or pleiotropy was observed in the analyses (P > 0.05).The stability of the findings was further confirmed through the funnel plot and the leave-one-out test, as depicted in Supplementary material 6.

DISCUSSION
This study presents the initial comprehensive assessment of the causal impacts of micronutrients and their supplements on PU.Through the utilization of two-sample MR analysis, we have established a definitive link between genetically predicted zinc deficiency and the development of PU.However, our analysis did not reveal a causal relationship between zinc supplements and PU.These results were consistently supported in sensitivity analysis.The implications of our findings offer valuable insights for further investigation, prevention, and management of PU.
The etiology of PU is not fully elucidated.Several studies 6,7,21,22 have demonstrated a potential association between aberrations in circulating micronutrients and PU, while conflicting findings 23,24 have been reported in other studies.Inconsistencies have also been observed in studies investigating the efficacy of micronutrient supplementation in the management of PU. 25,26 F I G U R E 2 Effect of micronutrients on PU in the MR analysis.Forest plot (A), scatter plot (B), leave-one-out plot (C), and funnel plot (D) of the causal effect of zinc on PU risk.(E) Forest plot of IVW analysis of 13 micronutrients on PU.
The limitations of observational studies, including susceptibility to confounding variables and reverse causality, as well as constraints in sample size, may introduce bias in the interpretations of previous research on micronutrients and PU.In this study, we utilized the robustness of MR analysis for confounder control and conducted a two-sample MR analysis to establish a more dependable causal association. 11Our investigation builds upon a recent retrospective cross-sectional study in Japan that demonstrated a significant correlation between zinc deficiency and PU and pressure zones, while our study further elucidates the causal relationship between zinc levels and the development of PU. 21In an experimental study involving animals, it was observed that zinc deficiency could potentially exacerbate vascular damage, oxidative stress, and apoptosis triggered by ischemia-reperfusion injury in the skin of mice.Additionally, a rise in ATP levels in the ischemia-reperfusion area was noted as a result of Langerhans cell depletion. 27This finding suggests a potential link between zinc deficiency and the development of PU, although further research is required to validate this hypothesis.
Prior research 6 has indicated a correlation between micronutrients, including selenium, copper, iron, and vitamin D, and the occurrence of PU.However, our study elucidates that there is no direct causal link between these micronutrients and PU.It is plausible that these micronutrients may impact PU indirectly by influencing other metabolites rather than being directly involved in the development of PU.
Numerous studies have presented conflicting results regarding the efficacy of zinc supplementation in the management of PU.A prospective study conducted in China demonstrated that the implementation of a nutritional support program significantly enhanced the healing of PU. 28 Furthermore, a recent comprehensive review by Saeg F 29 in 2021 concluded that the prognosis of patients with PU improved when treated with a combination of vitamin C and zinc.A systematic review and meta-analysis of clinical studies have provided evidence that zinc therapy is effective in promoting wound healing. 25Additionally, another systematic evaluation and meta-analysis found that the use of an arginine-, zinc-, and antioxidant-rich formula as an oral supplement and tube-feeding for a duration exceeding 8 weeks resulted in improved PU healing compared to a standardized formula. 30These findings underscore the beneficial impact of zinc supplementation in preventing PU, although conflicting results have been reported in some studies.In a clinical study conducted by Bafna K, it was observed that the routine administration of vitamins A and C, zinc, and arginine to patients with PU (stage IV) was not deemed justified. 31Furthermore, a narrative literature review on stress-induced skin lesions in children with critical illness found insufficient evidence to support a correlation between nutrition and skin health. 24Additionally, a review indicated that there is a lack of conclusive evidence supporting the efficacy of zinc and vitamin (A/C/D/E) supplementation in the prevention and treatment of PU. 26 A recent meta-analysis of 33 randomized controlled trials indicates that the efficacy of nutritional interventions in preventing and treating PU is inconclusive. 32The discrepancies in results may be attributed to the constraints of observational studies and inadequate sample sizes.Our study did not establish a definitive causal link between zinc supplementation and PU, casting doubt on the rationale for zinc use in future treatment of PU, further research is necessary to validate these findings.
This research employed MR to investigate the causal association between circulating micronutrients and their supplements and PU, highlighting its superiority over conventional observational studies in mitigating issues of reverse causation and confounding bias.Nevertheless, it is important to acknowledge certain limitations of the study.
Firstly, the exclusive inclusion of European descent participants in the GWAS study raises questions about the generalizability of the findings to diverse populations and geographic regions.Secondly, it should be noted that in the analysis of GWAS data of circulating micronutrients, micronutrient supplementation, and PU, a P-value threshold of <5 × 10 −8 was not employed, a standard threshold in the field. 17is omission may introduce bias into the results.Thirdly, the absence of comprehensive data on PU classification and micronutrient levels in patients with varying degrees of PU hindered the ability to explore the causal relationship between micronutrients and PU, this limitation could potentially introduce bias into the findings.Moreover, the restriction to a database lacking the capability for stratification by variables such as sex, age, diet, underlying disease, and severity of primary disease could have introduced a potential confounding bias.

CONCLUSION
Two-sample MR analysis was employed to investigate the potential causal association between micronutrients and supplements and the occurrence of PU.The results indicate a potential association between decreased zinc levels and the risk of PU, yet zinc supplementation did not demonstrate a significant reduction in the risk of PU formation.Further research is warranted to validate these findings and delve deeper into the role of zinc in the etiology, prevention, and management of PU.

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SNPs) strongly linked to micronutrients and their corresponding supplements as instrumental variables (IVs), a two-sample MR study was conducted to investigate the causal relationship between micronutrients and their supplements and PU.Three key assumptions were posited: Assumption 1, a strong association between the SNPs and the exposures (micronutrients and supplements); Assumption 2, the SNPs influenced the outcome solely through the exposure (micronutrients and supplements); and Assumption 3, the SNPs were not associated with confounding variables.14Figure 1 illustrates the flowchart of the entire analysis.
MR analyses were performed in the MR Base platform (App version: 1.4.3 8a77eb (October 25, 2020), R version: 4.0.3,Database version: 0.3.0(October 25, 2020), URL: http://app.mrbase.org/).MR analysis was conducted utilizing the inverse variance weighted (IVW) model, MR-Egger, Weighted median, Weighted mode, and Simple mode.The random effects IVW method was primarily employed due to its capacity to generate dependable estimates of causal effects, particularly in the context of heterogeneity.MR-Egger, Weighted median, Weighted mode, and Simple mode serve as supplementary approaches to enhance the reliability of the findings. 17To evaluate the variability of SNP effects related to micronutrients, their supplements, and PU, we conducted MR with inverse variance weighted (MR-IVW) analyses utilizing Cochran's Q statistic, as well as MR-Egger analyses employing Characteristics of the included studies.
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